SEROLOGY OF THE SOLUBLE ANTIGENS OF CLOSTRIDIUMPERFRINGENS TYPES A-F BY AGAR-GEL DIFFUSION

PAUL D. ELLNER AND CAROLYN D. BOHANDepartment of Medical Microbiology, College of Medicine, University of Vermont,

Burlington, Vermont

Received for publication August 4, 1961

ABSTRACT

ELLNER, PAUL D. (University of Vermont,Burlington) AND CAROLYN D. BOHAN. Serologyof the soluble antigens of Clostridium per-fringens types A-F by agar-gel diffusion. J.Bacteriol. 83:284-296. 1962.-A serological studyby agar-gel diffusion of the soluble antigens of39 strains of the Clostridium perfringens grouphas shown them to be extremely heterogeneous.Strain variation occurred within the six types,and common antigens shared among the sixtypes were frequently observed. Attempts toproduce type-specific sera by absorption wereunsuccessful, due to incomplete removal ofcommon antibodies.

Clostridium perfringens consists of a group ofrelated organisms, varying in source, patho-genicity, and toxin production, yet biochemicallyand morphologically indistinguishable.

Wilsdon (1931) proposed that the species bedivided into four types (A, B, C, D) based ontheir capacity to produce different toxins. How-ever, Wilsdon's studies were not based on puretoxins, and it remained for other workers (Glennyet al., 1933; Bosworth, 1943) to elucidate thenature and serological specificity of the majorlethal toxins produced by the various types ofthis organism. At the present time, six types of C.perfringens are recognized; these types (A, B, C,D, E, F) are based on the production of majorlethal toxins (Oakley, 1943; Oakley and Warrack,1953; Brooks, Sterne, and Warrack, 1957).The serological techniques which have been

utilized in efforts to evolve a classification of C.perfringens are varied. Orr and Reed (1940),using agglutination and precipitation reactions,found that only 6 of 85 cultures showed a closeantigenic relationship. With the exception of afew which displayed some overlapping of theirantigenic components, the remainder showed noantigenic relationship. Henderson (1940) based

his studies on the somatic, heat-stable 0 andheat-labile L antigens. He found the 0 antigensof type A strains to be strictly strain-specific,whereas the 0 antigen of type C strains wastype-specific. The 0 antigens of the type B strainsappeared to fall into two groups, whereas the 0antigens of type D exhibited a wide diversity.The L antigen appeared only in types B and Dand possessed little homogeneity. These resultswere confirmed by the work of Rodwell (1941),who found that the large number of subtypesnecessary for a classification of C. perfringensbased on agglutinin and agglutinin absorptionreactions would be impracticable.The use of the capsular polysaccharide as the

antigen for precipitation and agglutination testsindicated that a common polysaccharide existedfor most, if not all, strains of C. perfringens(Svec and McCoy, 1944).Bychenko and his associates (1959) established

65 serological groups of C. perfringens based onthe antigenic structure of the cells of varioustypes. They suggested that this large number ofgroups would make the practical use of thisprocedure for type determination rather difficult.

After the demonstration that double diffusionthrough agar gel could be utilized to studyantigenic components produced by C. perfringens,as well as other bacteria (Elek, 1949; Oakleyand Fulthorpe, 1953), the technique was used byBjorklund and Berengo (1954) to study thetoxins of various groups of clostridia. Orlans andJones (1958) used this method to study thesoluble antigens of types B, C, and D of C.perfringens.The present paper describes a study of the

relationships among the six types of C. per-fringens, without regard to their biologicalactivity, by means of the agar-gel method. Itshould be emphasized that, although the solubleantigens used may contain previously describedsoluble antigens (alpha, theta, kappa, etc.)usually designated as major lethal, minor lethal,

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and nonlethal antigens (Brooks et al., 1957),the preparations probably also contain othersoluble antigenic proteins that have not beencharacterized with respect to their biologicalactivity.

MATERIALS AND METHODS

Cultures. Cultures of C. perfringens werereceived from various sources (Table 1). Allstrains were tested for purity by streaking onlactose-egg yolk-milk agar (Willis and Hobbs,1959), and the species of typical isolated colonieswas confirmed by means of biochemical tests.All strains were maintained in spore medium(Ellner, 1956) until used. In all cases, liquid

TABLE 1. Sources of the 39 strains ofClostridium perfringens

Number Strain Source*

Type A12345678910111213141516

Type B1234

Type C12345

Type D123456

814HS-1S-1073895PB6K146AGSSR-1236243502Mf-1Mf-2Mf-312364

910603445

36736283716368712

15171946216743683

ahbbc

a

TABLE 1.-Continued

Number Strain Source*

Type E1 136 g2 1493 e3 1241 e4 140 g5 139 g

Type F1 3748 e2 143 g3 2076 e

* a, L. S. McClung, Indiana University; b, D.E. Dolby, Lister Institute of Preventive Medi-cine, London; c, A. Smith, University of Mary-land School of Medicine, Baltimore; d, H. Noyes,Walter Reed Army Institute of Research, Wash-ington, D.C.; e, M. Sterne, Wellcome ResearchLaboratories, Kent; f, R. H. Weaver, University ofKentucky, Lexington; g, H. W. Smith, AnimalHealth Trust, Essex; h, isolated from horsemanure; i, Walter Reed Army Institute of Re-search, Washington, D.C.; j, isolated from am-putation stump; k, isolated from a patient withsepticemia; 1, isolated from a patient after ampu-tation.

I media were boiled for 10 min and cooled priord to use. Cultures were incubated anaerobically,d either by the addition of a reducing substance to

the fluid medium or by incubation of plates in a

k Brewer jar.I Production of antigen. Trypticase soy brothf (10 ml) containing 0.1% sodium thioglycolatef and 0.5% agar was inoculated with the appro-f priate spore stock, and incubated at 37 C for

approximately 18 hr or until the culture wasg actively growing. This served as the inoculumg for the medium used for antigen productiong (Table 2). Five 200-ml bottles of antigen-produc-

ing medium (pH 7.2) were each inoculated withe 2.0 ml of the culture and incubated at 37 Cd for 15 hr. Cells were removed by centrifugatione at 12,000 X g for 15 min and the cleare supernatant fluid filtered through a type HAg membrane filter (Millipore Filter Corp., Bedford,

Mass.) to insure removal of all bacterial cells.g The soluble antigens were precipitated from theg filtrate by the addition of ammonium sulfate tog 70% saturation. The material which rose to theee top was skimmed off, dissolvred in 100 ml ofe distilled water, and dialyzed against buffered

saline (pH 6.0) containing 3.0% glycerol (as a

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TABLE 2. Composition of antigen-producing medium

Compound Final concentration

g/liter

Na2HPO4 6.95KH2PO4 2.46NZ Amine, type A* 20.0Thiotonet 20.0Glucose 10.0Sodium thioglycolate 1.0

mg/literCaCl2*2H20 10.0MnCl2 4H20 0.05CuSO4-5H20 0. 1ZnSO4g7H20 0.1MgSO4-7H20 150.0Fe2(S04)3 1.0Nicotinic acid 1.0Thiamine hydrochloride 1.0Pyridoxamine hydrochloride 1.0Calcium pantothenate 1.0Riboflavin 0.5Biotin 0.005Folic acid 0.005Adenine sulfate 10.0Uracil 10.0

* Sheffield Chemical, Norwich, N. Y.t Baltimore Biological Laboratory, Inc., Balti-

more, Md.

stablizing agent). Merthiolate (Eli Lilly & Co.,Indianapolis, Ind.) was added to a final concen-tration of 1:10,000, the protein content estimatedby measuring the optical density in a spectro-photometer at 260 and 280 m,u (Warburg andChristian, 1942), and the material stored at 5 C.This material constituted the soluble antigenicpreparation characteristic of each strain.

Immunization of animals. The soluble antigenicpreparations characteristic of each strain within atype were pooled and the protein concentrationof each of the six pools adjusted to 20 to 23 mgprotein/ml. A portion of each pool was treatedwith 0.15% formalin (Fulthorpe and Thomson,1960), incubated at 37 C for 72 hr, and stored at5 C for at least 10 days to destroy toxicity.White female rabbits weighing 2.5 to 3 kg

were immunized by a subcutaneous injection ofapproximately 50 mg of the formalin-treatedantigen emulsified (Berlin and McKinney, 1958)with Freund's incomplete adjuvant preparedfrom Arlacel A and Bayol F. A second injectionof 50 mg of the formalin-treated antigen adsorbed

on aluminum hydroxide gel was given subcuta-neously 14 days later.

After an additional 14 days, a rapid course ofapproximately 16 mg of the untreated antigenwas given by a series of four intravenous and twointraperitoneal injections over a 2-week period.This immunization procedure was shown toresult in high antibody titers. One week afterthe final injection (after trial bleedings) therabbits were bled to death. Three rabbits wereemployed for each antigen pool. The sera of thethree rabbits representing each antigen pool werecombined, preserved with Merthiolate (finalconcentration of 1:10,000), and frozen until use.

Agar-gel technique. The gel was prepared bydissolving 0.45 g of Jonagar #2 (Oxoid) in 100ml of 0.85% sodium chloride containing 0.01 %Merthiolate. The pH was adjusted to 7.2 to 7.4and the molten agar clarified by filtration throughWhatman no. 40 filter paper. After pouring intosiliconized petri plates, the gel was "aged" in therefrigerator for 3 days prior to use. Preliminarytests to determine the optimal concentration ofantigen and antiserum needed to produce themaximal number of lines indicated that concen-trations of antigen ranging from 0.5 to 10.0mg protein/ml were most suitable when usedwith undiluted serum. Wells were filled onlyonce with the appropriate antigen and sera to betested, and the plates were incubated in a moistchamber at room temperature for 1 to 4 days.Plates were examined daily. Agar plates werephotographed on the apparatus described byReed (1960), on Royal Ortho film (EastmanKodak Co., Rochester, N. Y.), exposed for 2.5 secat f/32, developed in DK 60a for 5 min at 20 C,and printed on Velour-R3 paper (Dupont)processed in Dektol (diluted 1:3).

Absorption studies. An attempt to producetype-specific sera was made by absorbing theserum with the pooled antigen of the remainingfive heterologous types rendered insoluble byprecipitation with aluminum hydroxide at pH7.0.

Preparation of pooled globulin. Equal volumesof each of the six antisera produced against thesix toxigenic types of C. perfringens were pooledand an equal volume of saturated ammoniumsulfate solution added. The resulting globulinprecipitate was collected by centrifugation,dissolved in a volume of 0.85% sodium chlorideequivalent to the original volume of the pooled

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sera, and dialyzed against 0.85% sodium chloride.Merthiolate was added to a final concentrationof 1:10,000, and the pooled globulin stored inthe freezer until used.

RESULTS

Strain variation within types. The solubleantigens of the sixteen type A strains were testedindividually with the antisera produced againstthe pooled type A antigens. The marked varia-tion existing among the antigens of the 16 strainsis shown in Fig. 1-6. The number of lines obtainedvaried from one to five, depending on the strain.When the same strains were tested with thepooled globulin, the number of lines varied fromone to seven.When testing the four type B strains against

antiserum produced against pooled type B anti-gens, lines varying in number from three to fourresulted (Fig. 7 and 8). The use of pooled globulinagainst the antigens of the type B strains in-creased the number of lines to five to six.

Following the same procedure, types C, D, E,and F were studied. The five strains of type C,when tested with type C antiserum, producedfour to six lines (Fig. 9 and 10), the numberincreasing to six to seven lines when pooledglobulin was employed. The number of linesproduced when the six type D strains weretested with type D antiserum was three to six(Fig. 11 and 12); six to seven lines resulted whenpooled globulin was used. The five type E strainsgave three to five lines with both homologousantiserum (Fig. 13 and 14) and pooled globulin.The three type F strains, when tested with typeF antiserum, produced four to five lines (Fig.15 and 16); with pooled globulin, four to six lineswere obtained. The results obtained when strainsrepresenting the various types were tested againstpooled globulin are shown in Fig. 17-23.

Strain relationship within types. From theabove, as well as from additional studies withhomologous antisera, it would appear that inmany cases strains within the same type are verysimilar, if not identical. Thus, of the 16 type Astrains, strains 3 and 8 appear identical, as do12, 13, 14, and 15. The remaining 11 strains oftype A differ. Of the four type B strains, strains1, 2, and 4 appear similar, but strain 3 differs.Two of the five type C strains, 4 and 5, appearsimilar; the remainder are different. Of the fivetype E strains, strains 1 and 5 appear similar,

but strains 2, 3, and 4 differ. None of the strainsin types D or F appeared identical.

Relationship between types. Pools of the solubleantigens of each type were prepared and testedagainst type A antiserum. Types B, C, D, E,and F antisera were tested in a similar mannerwith the pooled antigens of each type. Each ofthe six types possesses antigens in common withthe other types, regardless of the type of anti-sera used (Fig. 24-29). The same observationwas made when the six types were tested againstthe pooled globulin (Fig. 30). The antigenicrelationship between types B and D is alsoevident.

Although absorption of type A antiserum withthe pooled antigens of types B-F was not com-plete, the results obtained when this absorbedserum was tested against the pooled antigens ofeach of the six types indicate that there is noline unique to type A (Fig. 31).

Following the same procedure, type B anti-serum was absorbed with the pooled antigens oftypes A, C, D, E, and F. Figure 32 shows thatan antigen is shared with type C antigens.However, when type C antiserum was absorbed

with the pooled antigens of types A, B, D, E,and F, no antibody remained to react with anyof the six types, including type C itself. Thiswould indicate the absence of a unique antigenin type C.Type D antiserum was absorbed with the

pooled antigens of types A, B, C, E, and F.Results show the existence of an antigen uniqueto type D (Fig. 33). The antigen which appearedcommon to both types B and D was removedby the absorption of both types B and D anti-sera, thus confirming their presence in both types.

Results obtained after absorbing type E anti-serum with the pooled antigens of types A, B, C,D, and F, and type F antiserum with antigens oftype A-E, indicate the presence of a uniqueantigen in both types E and F (Fig. 34 and 35).

In an attempt to confirm the above absorptionexperiment, a sample of pooled globulin wasabsorbed with the pooled antigens of type A.In a similar manner, additional samples of theglobulin were absorbed with the pooled antigensof types B, C, D, E, and F, respectively. Each ofthe six "type-absorbed" globulin samples wastested against the six antigen pools. With A-absorbed globulin, two lines remained againstthe homologous antigen pool, with a correspond-

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FIG. 1 and 2. Type A strains 1-6 against type A antiserum. FIG. 3 and 4. Type A strains 6-11 againsttype A antiserum. FIG. 5 and 6. Type A strains 11-16 against type A antiserum.

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FIG. 7 and 8. Type B strains 1-4 against type B antiserum. FIG. 9 and 10. Type C strains 1-5 againsttype C antiserum. FIG. 11 and 12. Type D strains 1-6 against type D antiserum.

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17 18

FIG. 13 and 14. Type E strains 1-5 against type E antiserum. FIG. 15 and 16. Type F strains 1-3 againsttype F antiserum. FIG. 17. Some A and E strains against pooled globulin (PG). FIG. 18. Some B and Cstrains against pooled globulin (PG).

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FIG. 19. Some A and C strains against pooled globulin (PG). FIG. 20. Some D and E strains against pooledglobulin (PG). FIG. 21. Some C, D, and E strains against pooled globulin (PG). FIG. 22. Some A, D, E,and F strains against pooled globulin (PG). FIG. 23. Some A, E, and F strains against pooled globulin(PG). FIG. 24. Pooled antigens of types A-F against type A antiserum.

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FIG. 25. Pooled antigens of types A-F against type B antiserum. FIG. 26. Pooled antigens of types A-Fagainst type C antiserum. FIG. 27. Pooled antigens of types A-F against type D antiserum. FIG. 28. Pooledantigens of types A-F against type E antiserum. FIG. 29. Pooled antigens of types A-F against type F anti-serum. FIG. 30. Pooled antigens of types A-F against pooled globulin (US).

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FIG. 31. Pooled antigens oftypes A-F against typeA serum absorbed with B, C, D, E, andF antigens (AA).FIG. 32. Pooled antigens of types A-F against type B serum absorbed with A, C, D, E, and F antigens (AB).FIG. 33. Pooled antigens of types A-F against type D serum absorbed with A, B, C, E, and F antigens(AD). FIG. 34. Pooled antigens of types A-F against type E serum absorbed with A, B, C, D, and F anti-gens (AE). FIG. 35. Pooled antigens of types A-F against type F serum absorbed with A-E antigens (AF).FIG. 36. Pooled antigens of types A-F against pooled globulin absorbed with A antigen (-A).

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FIG. 37. Pooled antigens of types A-F against pooled globulin absorbed with B antigen (-B). FIG. 38.Pooled antigens of types A-F against pooled globulin absorbed with C antigen (- C). FIG. 39. Pooled anti-gens of types A-F against pooled globulin absorbed with D antigen (-D). FIG. 40. Pooled antigens of typesA-F against pooled globulin absorbed with E antigens (-E). FIG. 41. Pooled antigens of types A-F againstpooled globulin absorbed with F antigen (-F). FIG. 42. Pooled antigen of 39 strains (PA) and uninoculatedantigen-producing medium (M) against pooled globulin (PG).

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ing reduction in the number of lines obtainedwith the heterogenous antigen pools. In a likemanner, B-absorbed globulin gave one line; C-absorbed globulin gave one line; D-absorbedglobulin gave two lines; E-absorbed globulingave two lines; and F-absorbed globulin gavetwo lines (each when tested against the homolo-gous antigen pools). In every case, reduction of thenumber of lines against heterologous antigenpools was observed (Fig. 36-41).

Control studies. Results obtained when unin-oculated antigen-producing medium was testedagainst pooled globulin indicate that the mediumitself was antigenic. The two lines produced areprobably in response to the Thiotone and N-Z-Amine present in the medium.

These nonspecific lines have been taken intoconsideration in interpreting all data. The rela-tionship of these lines to lines formed against apool of the soluble antigens of all 39 strains canbe seen in Fig. 42.The pooled antigens of all 39 strains were

tested against rabbit antisera which had beenprepared against apparently unrelated antigens,such as ovalbumin, bovine y-globulin, bovineserum albumin, azo-bovine serum albumin, andEscherichia coli. Results showed these sera to becompletely devoid of antibodies which wouldcross-react with the clostridial antigens, and,consequently, indicates that C. perfringensantibody is normally absent in the rabbit.

DISCUSSION

It is apparent from these studies that thesoluble antigens of the C. perfringens group areextremely heterogeneous. Extreme strain varia-tion was observed within each of the six typesexamined. Moreover, many antigens are sharedamong the six types.Two lines of precipitation obtained in every

case were found to be nonspecific and due toantigenic components of the medium itself.Unless a medium for the production of bacterialantigens subsequently used to immunize animalshas been shown to be nonantigenic, such non-specific lines may be expected and might well bemisleading unless adequate controls areemployed.

After subtraction of the two nonspecific lines,type A, C, and D strains produced up to fivelines, type B and F strains up to four lines, andtype E strains up to three lines, when testedagainst the pooled globulin.

The increase in the number of lines usuallyobtained when pooled globulin was substitutedfor homologous sera might well be due to anadditive effect, in that antibodies which werebelow the level of precipitation in homologoussera, but common to several types, reached alevel in the pooled globulin which resulted in avisible line.Although absorption of homologous sera and

pooled globulin was not complete, neverthelessthe results of such experiments confirm thesupposition that there are antigens common totypes A through F. In addition, these experimentsstrongly suggest that there are no antigens uniqueto types A or C, whereas types D, E, and Fare each characterized by the possession ofunique antigens. Type B does not possess a uniqueantigen, as such, but demonstrates an antigenshared only with type C and another antigenshared only with type D.Attempts to correlate our findings with toxi-

genic types is very difficult, since typing is basedon the presence or absence of specific majorlethal, minor lethal, and nonlethal antigens,whereas the total soluble antigens in the presentstudy were utilized without regard to biologicalactivity. Orlans and Jones (1958), however, usingthe agar-gel diffusion method combined with theclassical tests for lethal, hemolytic, and enzy-matic factors, found that the antigens of thetype D strains conformed to the expected pattern,whereas type B and C strains showed widequalitative and quantitative variation. Theseworkers were able to identify precipitin bandsdue to alpha, beta, and epsilon toxins, but otherbands were present which were not identified.The relationship between types observed in

the present study does, however, show somesimilarity to intertype relationships demon-strated by the classical methods. Common anti-gens found among various types might wellrepresent alpha and nu toxins, common to alltypes; lambda toxin, common to types B, D, andE; beta and gamma toxins, common to types B,C, and F; theta toxin, common to all typesexcept F; kappa toxin, common to all typesexcept B (not including aberrant Iranian strains)and F; and mu toxin, common to all types exceptC and F. On the other hand, the common anti-gens could be due to capsular polysaccharidepresent in the cell-free filtrates and carried alongin the concentration procedure.The unique antigen of type E strains might be

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due to iota toxin, produced only by type E,whereas the unique antigens of types D and Fprobably represent an uncharacterized antigen.The antigen common to types B and C could bedue to delta toxin, produced only by these types.The antigen common to types B and D mightwell represent epsilon toxin, found only in thesetypes.The present study was undertaken to explore

the possibility of subelassifying the C. per-fringens group by means of the precipitationpatterns of their soluble antigens in agar gel.However, the extreme heterogeneity of thesomatic antigens found by other workers appar-ently extends to the soluble antigens as well, andthus renders this method impractical.

ACKNOWLEDGMENTS

The authors would like to express their ap-preciation to Elizabeth Brooks and Irene Battyof the Wellcome Research Laboratories for theirhelpful suggestions and critical evaluation of themanuscript.These studies were aided by a grant from the

National Fund for Medical Education.

LITERATURE CITED

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